Ex Vivo Confocal Microscopy: Revolution in Fast Pathology in Dermatology

J. Malvehy; J. Pérez-Anker; A. Toll; R. Pigem; A. Garcia; L.L. Alos; S. Puig

The British Journal of Dermatology. 2020;183(6):1011-1025.

Abstract and Introduction

Abstract

Confocal microscopy with in vivo and ex vivo modalities has been used in the evaluation of skin cancer and other dermatological disorders. Recent developments in ex vivo confocal microscopy allow for faster pathology assessment with greater accuracy by the visualization of cellular and architectural details, similarly to standard pathology, in either paraffin-embedded or frozen samples. They include the possibility of multimodal confocal microscopy using different lasers and fusion images. New staining protocols including immunostaining, with no damage to conventional histopathology preparation, have been recently described in melanocytic tumours and inflammatory skin diseases. Digital staining with haematoxylin and eosin is also incorporated in the new devices. In this review the applications of ex vivo confocal microscopy will be presented with the description of the technique and the technology, clinical evidence in dermatology and other fields, and further applications.

Introduction

In the last decade a revolution in dermatology has been produced with the introduction of confocal microscopy (CM). This is an optical method that in a few seconds can provide high-resolution images of in vivo tissue or fresh, nonfixed, thick pieces of specimens, which are optically scanned in slices, instead of using a microtome blade. Two different confocal imaging systems are currently available: (i) reflectance confocal microscopy (RCM), using the refractive properties of subcellular structures, and (ii) fluorescence confocal microscopy (FCM), using fluorochromes to increase the cell-to-stroma contrast. RCM with no contrast agents has been used mainly for in vivo accessible tissue examination in three main fields: the examination of retina and cornea,^[1–3] neoplastic and inflammatory skin diseases,^[4] and the exploration of body cavities reachable through probe-based confocal laser endomicroscopes.^[5–10]

Ex vivo CM has attracted the attention of dermatologists and Mohs surgeons in skin cancer, and also in other tumours including breast cancer, prostate cancer, nephrological tumours and central nervous system tumours. In dermatology, ex vivo CM has been applied mainly to surgical oncology and in particular to skin carcinoma for fast assessment of tumour margins during surgery in basal cell carcinoma (BCC), squamous cell carcinoma (SCC), dermatofibrosarcoma and some adnexal tumours. In high-risk tumours with frequent relapses after surgery, three-dimensional pathological evaluation (e.g. Mohs micrographic surgery, slow-Mohs) is recommended in international guidelines to ensure free margins for the management of skin cancer.^[11–13] Unfortunately, the availability of this procedure is limited in Europe due to the lack of reimbursement, the low number of dedicated units, and the elevated cost and time consumed with the procedure in busy referral hospitals, which are frequently overloaded with skin cancer interventions. Ex vivo CM can be seen as a technical solution due to the easy-to-use and faster methodology, which reduces the cost and surgical time.

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Table 1. Characteristics of confocal microscopy (CM) used in fresh tissue
System	Histolog	VivaScope2500M-G4
Modality of CM	FCM	FCM + RCM
Maximum image area	8 × 8 cm	2 × 2 cm
Laser	488 nm	785 nm/488 nm
Scanning characteristics	1600 × 1600 to 8000 × 8000 pixels	1024 × 1024 pixels
		Optical section thickness 4 μm
		Depth of scanning < 200 μm^a
		Magnification up to 540 ×
Display of images	Purple digital colour	Green (FCM); greyscale (RCM); fusion green–grey; H&E digital staining

FCM, fluorescence confocal microscopy; H&E, haematoxylin and eosin; RCM, reflectance confocal microscopy. ^aDepth of scanning of 200 μm is the limit of the optical resolution of the device. It may vary according to the optical characteristics of the tissue

Table 2. Comparison of *ex vivo* confocal laser microscopy (CLM) and conventional histopathology
	Ex vivo CLM (fresh tissue)	Histopath (frozen tissue)	Histopath (formalin-fixed tissue)
Staff	1	> 2: tissue preparation and tissue examination	> 2: tissue preparation and tissue examination
Technical requirements	Knife for sectioning. Liquids and recipients for stain procedure	Cryostat, microscope, stainer (optional)	Microtome, cooling plate, stretching table, water bath, incubator, microscope, stainer (optional)
Space	Minimal (1·5 m²)	5–10 m²	5–15 m²
Method of staining	Manual	Manual or automatic	Manual or automatic
Total time for section preparation (freezing and embedding, sectioning, staining, mounting on coverslip etc.)	1 min (RCM)	45 min (manual H&E)	17 h 38 min (manual H&E)
	3–7 min (FCM)	52 min (automated H&E)	17 h 45 min (automated H&E)
	2 h (manual IHC)	1 h 45 min (manual IHC)	18 h 55 min (manual IHC)
		2 h 30 min (automated IHC)	19 h 45 min (automated IHC)
Slide thickness	Lump tissue	8–10 μm	3–5 μm
Tissue reprocessing (additional staining)	Yes	No	No
Documentation	Direct digital images of the tissue (immediate telemedicine or reading in multiple facilities of the hospital)	Additional camera and computer or digital microscope needed for digital documentation. Telemedicine is not immediate	Additional camera and computer or digital microscope needed for digital documentation. Telemedicine is not immediate

Adapted from Hartmann et al.³⁵ FCM, fluorescence confocal microscopy; H&E, haematoxylin and eosin; IHC, immunohistochemistry; RCM, reflectance confocal microscopy.

Table 3. Staining methods for *ex vivo* confocal microscopy (CM) and application in dermatology
Single agent
Aluminium chloride, acetic acid, citric acid	RCM	Skin tumours^25–27
Acridine orange, methylene blue, toluidine blue, fluorescein, Nile blue or Patent Blue V	FCM	Skin tumours²²
Combinations
Methylene blue + toluidine blue	M-CM	Skin cancer¹⁴
Fluorescence proflavine + acetic acid + toluidine blue	FCM	Basal cell carcinoma³⁴
Acetic acid + acridine orange	M-CM	Basal cell carcinoma²³
Acridine orange + ethidium bromide	3-colour FCM	Basal cell carcinoma¹⁷
Immunostaining
FITC-labelled S-100A10, melan-A and anti-Ber-EP4 antibodies, NPs10@D1_ICF_Alexa647_DOTAGA Fe³⁺	FCM	Nonmelanocytic and melanocytic tumours^35,56
Fluorescent-labelled IgG and C3 antibodies	M-CM	Bullous pemphigoid³⁶
IgG, IgM, IgA, C3 and fibrinogen	M-CM	Cutaneous vasculitis³⁷

FCM, fluorescence confocal microscopy; FITC, fluorescein isothiocyanate; M-CM, multimodal confocal microscopy; RCM, reflectance confocal microscopy.

Table 4. Indications for *ex vivo* confocal microscopy in dermatology and other medical specialties
Fast diagnosis of skin tumours and assessment of skin cancer margins
Basal cell carcinoma,^24,43–47 squamous cell carcinoma,^{26, 45, 48–50} dermatofibrosarcoma,⁴¹ melanocytic lesions,^{35,51,52,54–56,77} tumours in special sites^55,62,78 (eyelid tumours, tumours of the conjunctiva, tumours of the nails)
Other tumours: breast, prostate, brain, thyroid, oesophagus, stomach, colon, lung, lymph node, cervix, oral mucosa and larynx^69–72
Diagnosis of infections: mucormycosis,⁶³ dermatophytosis,⁶⁴ herpes virus,⁶⁵ molluscum contagiousm,⁶⁶ aspergillosis⁷⁹
Inflammatory skin diseases^36,37,68 (vasculitis, bullous diseases, psoriasis, lichen planus, discoid lupus erythematosus)
Skin fillers⁶⁷

Table 5. *Ex vivo* confocal microscopy features described in basal cell carcinoma and squamous cell carcinoma in confocal mode
Basal cell carcinoma³²
1. Demarcated fluorescent areas with a higher nuclear density than the surrounding epidermis and adnexal structures (nuclear crowding) (FCM)
2. Peripheral palisading (peripheral polarized and aligned fluorescent cells) (RCM and FCM)
3. Clefting (black fluorescence-free area around the tumour mass) (FCM and RCM)
4. Nuclear pleomorphism (FCM)
5. Increased nucleus-to-cytoplasm ratio (RCM and FCM)
6. Modified stroma around the tumour (more densely nucleated dermis with fluorescent dots within a black background, corresponding to inflammatory cells and activated fibroblasts) (FCM and RCM)
Subtypes of BCC and features in ex vivo CM³²
Superficial BCC: tumour islands are connected to the epidermis and separated from the dermis by some clefts
Nodular BCC: tumour islands are larger and disconnected from the epidermis
Micronodular BCC: tumour islands are smaller
Infiltrative BCC: tumour islands are small, elongated and deeply located
Squamous cell carcinoma^49,50
1. Erosion/ulceration: 'black' sharply demarcated irregular areas in the epidermis and dermis, sometimes filled with amorphous material of varying brightness
2. Hyperkeratosis: grey to brightly shining layers causing increased thickness of upper layers of epidermis (corresponding to stratum corneum)
3. Parakeratosis: multiple layers of nucleated cells in stratum corneum
4. Architectural disarrangement: disarray of normal architecture of skin (epidermis, epidermal rete ridges, basal membrane, dermis and subcutis, including skin appendages)
5. Plump bright or speckled cells in the epidermis: bright roundish to polygonal cells with speckled appearance or indistinct borders in the epidermis and slightly larger size
6. Nest-like structures in the dermis: irregular aggregates of cells that are larger than inflammatory cells
7. Keratin pearls: bright, lamellar, sometimes speckled aggregations in the dermis corresponding to whorl-shaped keratin. Often a black centre of the structure is present
8. Peritumoral inflammatory infiltrate: roundish to oval, bright dots in the dermis
Poorly differentiated SCC exhibits few or absent keratin pearls and ulceration, and architectural and cytologic atypia are more prominent than in well-differentiated SCC

Table 6. Diagnostic accuracy of *ex vivo* confocal microscopy in skin cancer compared with traditional histopathology in different studies
Study	Device	Mode	Stain	Number, sections	Detection rates
Karen²⁴	V2000	FCM	AO	149 BCC Mohs submosaics	Se 96·6%, Sp 89·2%, PPV 92·3%, NPV 94·7%
Larson⁴³	V2000	FCM	AO	17 BCCs, 34 Mohs submosaics	Se 97%, Sp 89%, PPV 92%, NPV 95%
Bennàssar⁴⁴	V2500	FCM	AO	80 BCCs, 480 Mohs submosaics	Se 89%, Sp 99%, PPV 98%, NPV 97%
Mu⁴⁵	V2500	FCM	AO	64 BCCs/SCCs, 133 Mohs mosaics	Se 99%, Sp 93%
Espinasse⁴⁶	V2500	FCM	AO	42 BCCs of the eyelid	Se 100%, Sp 100%
Longo⁴⁷	V2500	FCM	AO	127 BCCs, 753 Mohs sections	Se 79·8%, Sp 95·8%, PPV 80·5%, NPV 95·7%
Peters³⁴	Histolog scanner	FCM	0·01% proflavine	148 suspected BCCs, 525 images (punch, shave/excision)	Se 73%, Sp 96%, 100% in Se and Sp in punch biopsies
Horn⁴⁸	V2500	FCM	AO	120 images, 10 SCCs	Se 95%, Sp 96%, PPV 96·3%, NPV 95·2%

AO, acridine orange; BCC, basal cell carcinoma; FCM, fluorescence confocal microscopy; NPV, negative predictive value; PPV, positive predictive value; SCC, squamous cell carcinoma; Se, sensitivity; Sp, specificity. V2000 and V2500, VivaScope 2000 and VivaScope 2500. These older versions of the VivaScope device do not have the capability to produce digital staining or fusion images.

Table 7. Limitations, reasons for lack of correlation with conventional histopathology and clues to solve this
False positives
• Hair follicles and eccrine glands or sebaceous glands may be confused with basal cell carcinoma islands. However, the former reveal no palisading, less fluorescence and smaller nuclei • Limited resolution with first-generation microscopes to distinguish the infiltrative cords of basal cell carcinoma from stroma. With new systems better resolution and multimodal modalities allow the identification of small tumour cells²³
False negatives
• Incorrect imaging of the sample due to the presence of artefacts and the inability to flatten the fresh tissue properly. This can be solved with proper training of the technician and use of new flattening devices^20,21 • Preparation of the specimen for standard histological examination and discarded outer sections of the tissue block from the classic paraffin-embedded and frozen sections. This outer tissue is not discarded in fresh-tissue examination with ex vivo confocal microscopy³⁴ • Recurrence rates for ex vivo confocal microscopy compared with conventional histopathology are unknown. Prospective studies are needed to determine the recurrence rates

Table 8. Characteristics of optical methods in *ex vivo* surgical pathology
Methods	Optical principle	Labelling^a	Cellular details	Images similar to H&E	Commercially available
OCT, FF-OCT, D-FF-OCT	Interferometry imaging by measuring the echo time delay and intensity of light that is backscattered from the tissue	Intrinsic	Only D-FF-OCT	No	Perimeter Medical Imaging, Toronto, ON, Canada; LLTech, Paris, France
SRS	Chemical contrast created by the vibrational properties of molecules caused by photons	Intrinsic	Yes	No	Invenio Imaging Inc., Santa Clara, CA, USA
PAM	Detection of (ultra)sound caused by light absorption in the tissue	Extrinsic	Yes	No	No
CM	Scanning point and filtering out-of-focus emitted light from the tissue through a pinhole conjugate to the focal plane	Intrinsic (RCM), extrinsic (FCM)	Yes	Yes	VivaScope 2500, Caliber Imaging and Diagnostics, Rochester, NY, USA; Histolog Scanner, SamanTree Medical SA, Switzerland
MUSE	Excitation of tissues with ultraviolet radiation to generate optically sectioned images	Extrinsic	Yes	Yes	No
SIM	Optical sectioning microscopy method that uses patterned fluorescence excitation	Extrinsic	Yes	Yes	No
LSM	Optical sectioning by rejecting out-of-focus light, using a thin selective illumination plane	Extrinsic	Yes	Yes	No
NLM	Nonlinear fluorescent excitation	Intrinsic and extrinsic	Yes	Yes	DermaInspect, JenLab GmbH, Jena, Germany

CM, confocal microscopy; D-FF-OCT, dynamic full-frame optical coherence tomography; FCM, fluorescence confocal microscopy; FF-OCT, full-field optical coherence tomography; LSM, light sheet microscopy; MUSE, microscopy using ultraviolet surface excitation; NLM, nonlinear microscopy (including multiphoton, two-photon and second-harmonic-generation microscopy); OCT, optical coherence tomography; PAM, photoacoustic microscopy; RCM, reflectance confocal microscopy; SIM, structured illumination microscopy; SRS, stimulated Raman spectroscopy. ^aLabelling refers to the use of intrinsic properties of the tissue or need of for extrinsic contrast (staining).

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Ex Vivo Confocal Microscopy: Revolution in Fast Pathology in Dermatology

Abstract and Introduction

Abstract

Introduction

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